Cell-specific modulation of papovavirus replication by tumor suppressor protein p53

Small DNA tumor viruses like human papillomaviruses, simian virus 40, and adenoviruses modulate the activity of cellular tumor suppressor proteins p53 and/or pRB. These viruses replicate as nuclear multicopy extrachromosomal elements during the S phase of the cell cycle, and it has been suggested that inactivation of p53 and pRb is necessary for directing the cells to the S phase. Mouse polyomavirus (Py), however, modulates only the pRB protein activity without any obvious interference with the action of p53. We show here that Py replication was not suppressed by the p53 protein indeed in all tested different mouse cell lines. In addition, E1- and E2-dependent papillomavirus origin replication was insensitive to the action of p53 in mouse cells. We show that in hamster (Chinese hamster ovary) or human (osteosarcoma 143) cell lines the replication of both Py and papillomavirus origins was efficiently blocked by p53. The block of Py replication in human and hamster cells is not caused by the downregulation of large T-antigen expression. The deletion analysis of the p53 protein shows that the RPA binding, proline-rich regulatory, DNA-binding, and oligomerization domains are necessary for p53 action in both replication systems. These results indicate that in mouse cells the p53 protein could be inactive for the suppression of papovavirus replication.     

A set of stage-dependent embryonic antigens expressed in cell cultures of BALB/c mouse embryos and in transformed cell lines

A rabbit antiserum (A2) directed against the detergent-solubilized fraction of the simian virus 40-transformed mouse embryo fibroblast cell line VLM detects common antigens in primary cell cultures from BALB/c mouse embryos and in transformed cell lines from various species. Positively reacting cell cultures show a set of polypeptides with molecular weight species p86, p74, p68, p46, p42, p40, and p35. As tested by Western blotting procedures, all immunoprecipitated proteins carry immunologically reactive determinants. By analysis with two-dimensional gel electrophoresis, all precipitated polypeptides show charge heterogeneities. Concerning the two major members of the protein set, p40 consists of at least four subspecies with isoelectric points in the range of pH 6.2-6.8, whereas p35 is composed of two subspecies focusing between pH 6.4 and pH 7.2. By comparison of the two-dimensional patterns of p35 of various transformed cell lines, a basic (pH 6.6-7.2) and an acidic (6.4-6.6) charge type of p35 could be observed. Comparative analyses of primary cell cultures from 12-16-day mouse embryos show the immunoprecipitated set of polypeptides only in the 16-day embryo cell cultures. After six further propagations, these cells express the immunoreactive proteins as strongly as the primary cell cultures. In embryonic cell cultures of day 14 of gestation the expression of this set of antigens is induced only when cells are propagated at least six times. Under identical conditions these proteins could not be induced in cell cultures of 18-day-old mouse embryos. None of the polypeptides could be immunoprecipitated from primary mouse kidney cell cultures of 12-day-old mice even when the cultures were propagated at least 15 times. This set of polypeptides is also present in simian virus 40-transformed cells of hamster, rat, monkey, and human origin. These findings suggest that in simian virus 40-transformed mouse cells, in addition to p53, the synthesis of other embryonic antigens is reactivated. The presence of the described set of polypeptides in polyoma virus-transformed cells of rat and mouse origin and in cell lines derived from malignant human tumors might indicate common functions in metabolic patterns of transformed cells.     

The p53 status of Chinese hamster V79 cells frequently used for studies on DNA damage and DNA repair.

Chinese hamster lung fibroblast V79 cells have been widely used in studies of DNA damage and DNA repair. Since the p53 gene is involved in normal responses to DNA damage, we have analyzed the molecular genetics and functional status of p53 in V79 cells and primary Chinese hamster embryonic fibroblast (CHEF) cells. The coding product of the p53 gene in CHEF cells was 76 and 75% homologous to human and mouse p53 respectively, and was 95% homologous to the Syrian hamster cells. The V79 p53 sequence contained two point mutations located within a presumed DNA binding domain, as compared with the CHEF cells. Additional immunocytochemical and molecular studies confirmed that the p53 protein in V79 cells was mutated and nonfunctional. Our results indicate that caution should be used in interpreting studies of DNA damage, DNA repair and apoptosis in V79 cells.     

Characterization of p53 in Chinese hamster cell lines CHO-K1, CHO-WBL, and CHL: implications for genotoxicity testing.

Since the p53 gene function is critical to how a cell responds to DNA damage, we investigated the p53 status in Chinese hamster cell lines commonly used in genotoxicity tests for cytogenetic damage around the world. These included: Chinese hamster ovary K1 (CHO-K1), Chinese hamster ovary WBL (CHO-WBL), and Chinese hamster lung (CHL) cells. The results of DNA sequencing, protein analysis, and cell cycle analysis demonstrate that the CHO-K1 and CHO-WBL cell lines have mutant p53 sequence [a mutation in codon 211 in exon 6 resulting in a change from Thr (ACA) to Lys (AAA)], mutant protein (high spontaneous levels that are non-inducible after X-irradiation), and mutant function (lack of G1 checkpoint). Interestingly, the CHL cell line has a completely wild-type p53 DNA sequence. However, the CHL cells have an abnormally high spontaneous level of wild-type p53 protein expression that is not inducible after X-irradiation, yet there is some evidence of G1 delay after irradiation. The protein data suggests that p53 in CHL cells is not being regulated normally, and thus is probably not functioning normally. The mechanism leading to this abnormal regulation of p53 in CHL cells clearly does not involve mutation in the p53 gene. Overall, the CHL cell line may be similar to the CHO cell lines, in that they all appear to have abnormal p53 function. Further work is needed to determine whether the presence of spontaneously high levels of wild-type p53 in CHL cells results in a difference in response to DNA damage (quantitatively or qualitatively) compared to the p53 mutant CHO cell lines.     

Assignment of genes encoding metallothioneins I and II to Chinese hamster chromosome 3: evidence for the role of chromosome rearrangement in gene amplification.

Cadmium resistant (Cdr) variants with coordinately amplified metallothionein I and II (MTI and MTII) genes have been derived from both Chinese hamster ovary and near-euploid Chinese hamster cell lines. Cytogenetic analyses of Cdr variants consistently revealed breakage and rearrangement involving chromosome 3p. In situ hybridization with a Chinese hamster MT-encoding cDNA probe localized amplified MT gene sequences near the translocation breakpoint involving chromosome 3p. These observations suggested that both functionally related, isometallothionein loci are linked on Chinese hamster chromosome 3. Southern blot analyses of DNAs isolated from a panel of Chinese hamster X mouse somatic cell hybrids which segregate hamster chromosomes confirmed that both MTI and MTII are located on chromosome 3. We speculate that rearrangement of chromosome 3p could be causally involved with the amplification of MT genes in Cdr hamster cell lines.     

Role of the p53 protein in cell proliferation as studied by microinjection of monoclonal antibodies.

Two monoclonal antibodies against the p53 protein, PAb 122 and 200-47, were microinjected into mammalian cells as a probe to determine the role of the p53 protein in cell proliferation. PAb 122 recognizes the p53 proteins of mouse and human cells but not of hamster cells, whereas 200-47 recognizes the p53 proteins of mouse and hamster cells but not of human cells. The ability of these antibodies to inhibit serum-stimulated DNA synthesis of cells in culture correlates with their ability to recognize the species-specific antigenic determinants. More important, however, is the observation that microinjected PAb 122 inhibits the transition of Swiss 3T3 cells from G0 to S phase, but has no effect on the progression of these cells from mitosis to the S phase.     

Chromosomal localization of the Moloney sarcoma virus mouse cellular (c-mos) sequence.

The Moloney sarcoma virus-specific onc gene, referred to as v-mos, was used as probe to hybridize to restricted DNAs from various mouse-Chinese hamster hybrid cell lines. These hybrid cells contain, in addition to all of the Chinese hamster chromosomes, various numbers (less than a full complement) of mouse chromosomes. Comparison of the presence or absence of the mouse cellular mos gene with the known karyotype in each of the hybrid cell lines allows us to conclude that the mos gene is on mouse chromosome 4.     

Diminished G1 checkpoint after gamma-irradiation and altered cell cycle regulation by insulin-like growth factor II overexpression

High levels of insulin-like growth factor II (IGFII) mRNA expression are detected in many human tumors of different origins including rhabdomyosarcoma, a tumor of skeletal muscle origin. To investigate the role of IGFII in tumorigenesis, we have compared the mouse myoblast cell line C2C12-2.7, which was stably transfected with human IGFII cDNA and expressed high and constant amounts of IGFII, to a control cell line C2C12-1.1. A rhabdomyosarcoma cell line, RH30, which expresses high levels of IGFII and contains mutated p53, was also used in these studies. IGFII overexpression in mouse myoblast C2C12 cells causes a reduced cycling time and higher growth rate. After gamma-irradiation treatment, C2C12-1.1 cells were arrested mainly in G0/G1 phase. However, C2C12-2.7 and RH30 cells went through a very short G1 phase and then were arrested in an extended G2/M phase. To verify further the effect of IGFII on the cell cycle, we developed a Chinese hamster ovary (CHO) cell line with tetracycline-controlled IGFII expression. We found that CHO cells with high expression of IGFII have a shortened cycling time and a diminished G1 checkpoint after treatment with methylmethane sulfonate (MMS), a DNA base-damaging agent, when compared with CHO cells with very low IGFII expression. It was also found that IGFII overexpression in C2C12 cells was associated with increases in cyclin D1, p21, and p53 protein levels, as well as mitogen-activated protein kinase activity. These studies suggest that IGFII overexpression shortens cell cycling time and diminishes the G1 checkpoint after DNA damage despite an intact p53/p21 induction. In addition, IGFII overexpression is also associated with multiple changes in the levels and activities of cell cycle regulatory components following gamma-irradiation. Taken together, these changes may contribute to the high growth rate and genetic alterations that occur during tumorigenesis.     

Species-specific differences in the toxicity of rhodamine 123 towards cultured mammalian cells

The toxicity of cationic fluorescent dye, rhodamine 123, towards a number of independently established cell lines from three different species, namely human, mouse, and Chinese hamster, has been examined. All of the cell lines from any one species that were examined were found to exhibit similar sensitivities towards rhodamine 123 and no appreciable differences were observed between the normal and transformed cell types. However, in comparison to the cells of human origin, mouse and Chinese hamster cell lines exhibited about 10-fold and 70-fold higher resistance, respectively, and these differences appeared to be species related. In contrast to rhodamine 123, no differences in relative toxicities for these cell lines were observed for the structurally related neutral dye, rhodamine B. Fluorescence studies with rhodamine 123 show that in comparison to mouse and Chinese hamster cells, the more sensitive human cells show much higher uptake/binding of the drug, and a good correlation was seen in these studies between the extent of dye uptake/binding and the relative sensitivities of cell lines to rhodamine 123. These results provide evidence that the observed species-related differences in cellular toxicities are due to differences in the cellular uptake/binding of the dye.     

Structural analysis of an RNase T1 variant with an altered guanine binding segment

Information concerning the function of recombination proteins in mammalian cells has been obtained from biochemical studies, but little is known about their mechanisms of action in growing cells. The eukaryotic recombination protein RAD51, a homologue of the Escherichia coli RecA protein, has been shown to interact with various proteins, including the p53 protein, the guardian of genomic stability maintenance. Here, the hamster RAD51 protein, CgRAD51, has been overexpressed in the SPD8 cell line, derived from Chinese hamster V79 cells. This cell line offers unique possibilities for studying different mechanisms for homologous recombination on endogenous substrates. We report that the SPD8 cell line contains a mutated p53 gene, which provides new insights into the recombination process in these cells. The present study demonstrates that overexpression of CgRAD51 in these cells results in a two- to threefold increase in endogenous recombination. In addition, sequence analysis indicated that RAD51 promotes homologous recombination by a chromatid exchange mechanism.     

Species specific differences in the toxicity of mithramycin, chromomycin A3, and olivomycin towards cultured mammalian cells

Three structurally related anticancer drugs, mithramycin, chromomycin A3, and olivomycin, showed large unexpected differences (up to more than 1000 fold) in their toxicity towards cultured cells from various species (human, Chinese hamster, Syrian hamster, and mouse). Among the cell types examined, human cells (both a diploid fibroblast cell strain and HeLa cells) were maximally sensitive to all these drugs, followed by the Syrian hamster kidney cells (BHK 21). The mouse (LMTK- cells) and Chinese hamster (CHO) cells, which were more resistant, showed interesting differences in their sensitivity towards these drugs. For example, whereas the mouse cells were more resistant to mithramycin than CHO cells, the sensitivity pattern was reversed for both chromomycin A3 and olivomycin. In cell extracts derived from human, mouse, and Chinese hamster cells RNA synthesis, which is the cellular target of these drugs, showed identical sensitivity to both mithramycin and chromomycin A3, indicating that the species specific differences in the toxicity to these drugs are at the level of cellular entry of these compounds. Based on the structures of these glycosidic antibiotics and their patterns of toxicity, it is suggested that the intracellular transport of these drugs involves specific interactions between the sugar residues on these compounds and some type of cell surface receptor(s), which differ among different cell types. Some implications of these results for toxicity studies are discussed.     

A scaffold for the Chinese hamster genome

Chinese hamster ovary (CHO) cells are a prevalent tool in biological research and are among the most widely used host cell lines for production of recombinant therapeutic proteins. While research in other organisms has been revolutionized through the development of DNA sequence-based tools, the lack of comparable genomic resources for the Chinese hamster has impeded similar work in CHO cell lines. A comparative genomics approach, based upon the completely sequenced mouse genome, can facilitate genomic work in this important organism. Using chromosome synteny to define regions of conserved linkage between Chinese hamster and mouse chromosomes, a working scaffold for the Chinese hamster genome has been developed. Mapping CHO and Chinese hamster sequences to the mouse genome creates direct access to relevant information in public databases. Additionally, mapping gene expression data onto a chromosome scaffold affords the ability to interpret information in a genomic context, potentially revealing important structural and regulatory features in the Chinese hamster genome. Further development of this genomic scaffold will provide opportunities to use biomolecular tools for research in CHO cell lines today and will be an asset to future efforts to sequence the Chinese hamster genome.     

The gene and the pseudogene for mouse p53 cellular tumor antigen are located on different chromosomes.

The chromosomal assignments of the two genes encoding the murine p53 cellular tumor antigen were determined by using a panel of mouse-Chinese hamster somatic cell hybrid clones and a mouse p53-specific cDNA clone. One gene, probably the functional member of the family, was found to be on chromosome 11. The other gene, which is probably a processed pseudogene, was assigned to chromosome 14. The potential relevance of these findings to documented cases of chromosome 11 trisomy are also discussed.     

Variation in antigenic determinants of p53 transformation-related protein obtained from various species

p53 is a cellular-encoded transformation-related protein. It is synthesized at elevated levels in tumor cells but has also been detected at low concentrations in several types of nontransformed cells. The p53 of tumor cells is immunogenic and elicits specific antibody production. The antigenic determinants of the p53 protein were studied by specific binding to anti-p53 monoclonal antibodies obtained from the RA3-2C2, PAb122, and PAb421 established hybridoma cell lines, and their conservation was followed in various animal species. We found that whereas mouse p53 efficiently immunoprecipitated with all three anti-p53 monoclonal antibodies, human and rat p53 bound PAb122 and PAb421 but lacked a determinant binding RA3-2C2. The hamster p53 molecule represented a third category, which immunoprecipitated with polyclonal anti-p53 antibodies but failed to bind all three monoclonal antibodies analyzed here. Using these monoclonal antibodies, we detected no variations between p53 found in transformed and p53 found in nontransformed cells, within a given species. The results also showed that RA3-2C2, which recognizes a mouse-specific determinant, binds a site located at a proteolytic digestion fragment of the p53 molecule that differs from that containing PAb122 and PAb421 recognition site(s). p53 is a single protein that can be immunoprecipitated through different antigenic determinants that vary between species.     

Characterization of the cell of origin for small cell lung cancer

Small cell lung carcinoma (SCLC) is a neuroendocrine subtype of lung cancer that affects more than 200,000 people worldwide every year with a very high mortality rate. Here, we used a mouse genetics approach to characterize the cell of origin for SCLC; in this mouse model, tumors are initiated by the deletion of the Rb and p53 tumor suppressor genes in the lung epithelium of adult mice. We found that mouse SCLCs often arise in the lung epithelium, where neuroendocrine cells are located, and that the majority of early lesions were composed of proliferating neuroendocrine cells. In addition, mice in which Rb and p53 are deleted in a variety of non-neuroendocrine lung epithelial cells did not develop SCLC. These data indicate that SCLC likely arises from neuroendocrine cells in the lung.Key words: Rb, p53, SCLC, cell of origin, cancer, lung, neuroendocrine     

Co-dominant markers for the selection of somatic dihybrids and polyhybrids

Resistance to ouabain and puromycin are shown to represent very useful co-dominant characters for the selection of somatic hybrids between mammalian cells, after fusion with polyethylene glycol. We therefore used, with success, a number of Chinese hamster and mouse cell lines carrying these markers in association with thymidine kinase and hypoxanthine-guanine-phosphoribosyl transferase deficiency for selection of hybrids of triparental origin.     

Stable formation of mutated p53 multimers in a Chinese hamster cell line causes defective p53 nuclear localization and abrogates its residual function

We have previously described a methotrexate-resistant cell line (MTX M) characterized by amplified dihydrofolate reductase (DHFR) genes, cytoplasmic p53 localization, and p53 stable tetramers. To investigate the p53 functionality in MTX M, the effect of chemical/physical agents was studied. In MTX M cells, DNA damage did not induce p53 or mdm-2 protein, while in the parental V79 cells, a residual p53 activity was found. cDNA sequencing showed that V79 and MTX M cells share the same mutations, indicating that the complete loss of p53 function in MTX M cells was due to cytoplasmic sequestration of a mutated p53 with residual activity. In Chinese hamster, both p53 and DHFR genes map on short arm of chromosome 2 suggesting that p53 itself might be amplified. However, fluorescence in situ hybridization with a hamster p53 probe showed only a single signal. Thus, the presence of p53 stable tetramers in MTX M cells, although correlated with DNA amplification, could not be the consequence of either p53 or DHFR gene amplification. Expression of a C-terminal human p53 peptide does not induce p53 nuclear accumulation, indicating that the cytoplasmic localization is due to a mechanism different from that already described in cancer cell lines. Treatments with Sodium Butyrate induced beta-tubulin polymerization, but did not apparently organize a normal microtubule network, which is shown to be important for the p53 localization. Our data indicated that in MTX M cells, p53 is sequestered in the cytoplasm by a novel mechanism that abrogates p53 residual function.     

Conservation of the syntenic group enol - pgd - pgm in mammals: its assignment to chromosome 2 in the Chinese hamster

Hybrids between cells from mouse permanent lines and Chinese hamster thymus cells explanted from animals maintained mouse chromosomes and lost most hamster chromosomes. In twenty-seven hybrids examined for expression of enolase 1. phosphogluconate dehydrogenase, and phosphoglucomutase, the Chinese hamster forms of the three enzymes were either expressed together, or not expressed at all. Thus, the three genes eno1, pgd, and pgm appear syntenic in Chinese hamster as they are in man (chromosome 1p), and in mouse (chromosome 4). The three markers map on the Chinese hamster chromosome 2.     

p53-independent apoptosis is induced by the p19ARF tumor suppressor

p19(ARF) is a potent tumor suppressor. By inactivating Mdm2, p19(ARF) upregulates p53 activities to induce cell cycle arrest and sensitize cells to apoptosis in the presence of collateral signals. It has also been demonstrated that cell cycle arrest is induced by overexpressed p19(ARF) in p53-deficient mouse embryonic fibroblasts, only in the absence of the Mdm2 gene. Here, we show that apoptosis can be induced without additional apoptosis signals by expression of p19(ARF) using an adenovirus-mediated expression system in p53-intact cell lines as well as p53-deficient cell lines. Also, in primary mouse embryonic fibroblasts (MEFs) lacking p53/ARF, p53-independent apoptosis is induced irrespective of Mdm2 status by expression of p19(ARF). In agreement, p19(ARF)-mediated apoptosis in U2OS cells, but not in Saos2 cells, was attenuated by coexpression of Mdm2. We thus conclude that there is a p53-independent pathway for p19(ARF)-induced apoptosis that is insensitive to inhibition by Mdm2.     

Cloning and characterization of Chinese hamster p53 cDNA

We have cloned and sequenced Chinese hamster p53 cDNA and have compared the p53 sequence in different Chinese hamster cell lines to several relevant phenotypes. Our results indicate that a mutation in CHO cells that changes Thr²¹¹ to Lys²¹¹ abrogates the ability to arrest in G₁ and apparently renders cells capable of amplifying DNA. However, this mutation has no effect on the G₂ checkpoint or on acute down-regulation of DNA replication after a radiation challenge.